G protein-coupled-receptors (GPCRs) constitute the largest family of integral membrane proteins. More than 800 GPCRs have been identified, the underlying genes represent 2 to 3% of the coding sequences in the human genome. They participate in the regulation of most physiological functions and are the targets of approximately 30% of currently marketed drugs. Among GPCRs, the arginine-vasopressin V2 receptor subtype (also known as vasopressin-2, vasopressin 2, vasopressin type 2 or V2 receptor and abbreviated AVPR2 or V2R) is considered as a prototype of receptors coupled to the intracellular cyclic adenosine monophosphate (cAMP) signalling pathway. It represents a fundamental and therapeutic model.
Arginine-vasopressin (AVP), also known as anti-diuretic hormone, is a 9-amino acid cyclic peptide produced in the hypothalamus. It exerts its action via 3 distinct receptor subtypes: vasopressin 1a (V1aR), vasopressin 1b (V1bR) and vasopressin 2 (V2R). The actions of V1aR and V1bR are mediated via their interaction with the G protein Gq leading to an increase in intracellular calcium concentration. The V1aR is mainly expressed in the liver, smooth muscle cells (vasoconstriction), platelets, brain, retina and reproductive organs, whereas the V1bR is mainly expressed in the anterior pituitary gland where its stimulation controls the corticotropic axis in response to stress. V1bR is also expressed in the pancreas and adrenal glands where it regulates the secretion of glucagon, insulin and catecholamines. The V2R is located in the renal collecting duct where it is coupled to the Gs protein. Gs activate adenylate cyclase which in turn leads to an increased cyclic AMP production. cAMP activates protein kinase A which phosphorylates and activates water channels called aquaporins responsible for water reabsorption in the kidney. The V2R is also expressed in the inner ear where it regulates its osmotic pressure.
V2R participates in the regulation of major physiological functions and is a target for the development of new therapeutic drugs for treating various pathologies. Non-peptidic V2R antagonists, named vaptans, have been developed and tested in numerous clinical trials but most of them were not approved by the drug authorizing authorities because of their hepatotoxicity. Today, Tolvaptan (OPC-41061) and Conivaptan (YM-087) only, have been approved by the FDA, and/or EMEA.
Pathological Conditions Characterized by Euvolemic or Hypervolemic Hyponatremia
The excess secretion of AVP is a key etiologic factor for diseases such as hyponatremia and explains why the use of V2R antagonists is so efficient in pathological conditions characterized by euvolemic or hypervolemic hyponatremia, such as SIADH (Syndrome of Inappropriate AntiDiuretic Hormone secretion), hepatic cirrhosis and congestive heart failure (CHF; Ghali et al., Cardiology, 2008, 111, 147-157). SIADH is caused by the hypersecretion of AVP which leads to excessive water retention and consequently a decrease in Na+ concentration and edema in the lung and central nervous system. By their diuretic effect V2R antagonists increase or normalize serum Na+ levels. Tolvaptan (OPC-41061) and Conivaptan (YM-087) have been approved by the FDA, and/or EMEA on patients diagnosed with euvolemic and hypervolemic hyponatremia, SIADH, congestive heart failure and cirrhosis (Arai et al., Curr Opin Pharmacol., 2007, 7, 124-129; Manning et al., Prog. Brain Res., 2008, 170, 473-412). V2R antagonists could be potentially beneficial in other pathological conditions characterized by euvolemic or hypervolemic hyponatremia, such as cerebral oedema (Walcott et al., Neurotherapeutics, 2012, 9, 65-72).
Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD)
The NSIAD is due to V2R mutations such as R137C and R137L which are responsible for a constitutive activity of the receptor. Patients present hyponatremia and a high urinary osmolality despite low serum vasopressin levels. The V2R antagonists Satavaptan and Tolvaptan, either in cell cultures (Tenenbaum et al., PLoS One, 2009, 4, e8333) or in patients with NSIAD (Decaux et al., JASN, 2007, 18, 606-612) are unable to inhibit this constitutive activity.
Congenital Nephrogenic Diabetes Insipidus (cNDI)
This disease is associated with inactivating mutations of the V2R. Deficient receptors are sequestered inside the cell and cannot be reached by circulating AVP. This leads to polyuria with severe dehydration in particular in children. The V2R antagonists (vaptans) behave as pharmacochaperones, they are able to penetrate the cell and can rescue the mutant receptors (Morello et al., J. Clin. Investigation, 2000, 105, 887-895). In some cases this allows the receptor to be stimulated by AVP in order to restore the antidiuretic effect (Bernier et al., J. Am. Soc. Nephrol., 2006, 17, 232-243; Robben et al., Am. J. Physiol. Renal. Physiol., 2007, 292, 253-260). Vaptans were tested in clinical trials but most of them were not approved by the FDA because of their hepatotoxicity (Manning et al., Prog. Brain Res., 2008, 170, 473-512). Today, Tolvaptan only, has been approved by the FDA.
Polycystic Kidney Disease
Polycystic kidney disease is characterized by the appearance of numerous cysts which ultimately lead to end-stage renal failure in the majority of patients. Patients with mutations in the PKD1 and PKD2 genes are unable to concentrate urine despite having a high vasopressin concentration in the blood. The current available treatments for this pathology are dialysis or transplantation. V2R antagonists were shown to slow down the course of the disease in different animal models of recessive and dominant polycystic kidney disease including the CD1pcy/pcy mouse model (Gattone et al., Nature Medicine, 2003, 9, 1323-1326; Torres, V. E., Clin. J. Am. Soc. Nephrol., 2008, 3, 1212-1218; Wang et al., J. Am. Soc. Nephr., 2008, 19, 102-108). The CD1pcy/pcy mouse is a model for autosomal recessive cystic kidney disease that is caused by a missense mutation in the NPHP3 gene which encodes nephrocystin-3, a protein involved in renal tubular development and function. This mutation leads to renal cyst formation and end-stage renal failure. Moreover, phase III clinical trials on patients with autosomal dominant polycystic kidney disease showed that Tolvaptan administered to patients during 3 years decreased proliferation of cyst-lining epithelial cells (Higashihara et al., Clin. J. Am. Soc. Nephrol., 2011, 6, 2499-2507).
Cancer
By interacting with arrestin V2R stimulation leads to the activation of signaling pathways involving cAMP and MAP kinase thus favoring a proliferative response. For example, AVP injection in the rat induces the proliferation of renal tubular epithelial cells that can be inhibited by V2R antagonists (Alonso et al., Endocrinology, 2009, 150, 239-250). Moreover, diuretics such as V2R antagonists were able to inhibit the proliferation of renal cancer cells (Bolignano et al., Urol. Oncol., 2010, 28, 642-647) and of pulmonary cancer cells (Pequeux et al., Endocr. Relat. Cancer, 2004, 11, 871-885. These results indicate that V2R antagonists are good therapeutic candidates against different types of cancer.
Thrombosis
The Von Willebrand factor (VWF) is implicated in primary hemostasis. It has been demonstrated that AVP and also the V2R specific agonist, dDAVP (Minirin®), increases VWF and factor VIII levels via their interaction with V2R (Kaufmann et al., J. Clin. Invest., 2000, 106, 107-116). An excess in coagulation can lead to thrombosis (clots) which could be cured by the use of V2R antagonists thus limiting the secretion of coagulation factors.
Ménière Disease
In France, the incidence of Ménière disease is estimated to be 1/13,300. Its pathogenesis is largely unknown. Endolymphatic swelling is considered a pathognomonic sign. It may result either from endolymph hypersecretion or from insufficient reabsorption. Menière pathology of the inner ear seems to be the origin for symptoms such as vertigo, nausea, tinnitus and hearing loss. It is believed that the increased pressure in the cochlea compromises the ability of cilial cells to detect correctly the soundwave or movements (Kitahara et al., J. Neuroendocrinol., 2008, 20, 1295-1300). Presently, treatment with acetazolamide (diamox) is used which inhibits carbonic anhydrase and acts as hypokaliemic diuretic. By reducing the osmotic pressure into the inner ear, V2R could be used for treating Ménière disease.
Although vaptans have clearly proven the therapeutic effect of V2R antagonists on various pathologies, their therapeutic use is limited by some major drawbacks:                Vaptans are hepatotoxic due to their inhibitory effect on cytochrome CYP3A4. For this reason, their use necessitates a tight monitoring of the patients and their long-term administration is limited. Some of them are only injected intravenously which restrict their use to hospitalized patients.        Vaptans have some selectivity only for V2R with V2/V1a selectivity index which varies from 112 (Satavaptan) to 0.15 (Conivaptan).        Vaptans are agonists for MAP kinase activation. Therefore, they are unable to block completely specific V2R-associated signaling pathways.        Vaptans are poorly soluble in physiological buffers and have limited bioavailability (Bernier et al., JASN, 2006, 17, 591-).        
Therefore, there is a need for new V2R antagonists with improved properties for therapeutic use, particularly with increased V2R selectivity and reduced toxicity, compared to currently available non-peptidic V2R antagonists.
Kunitz domains are the active domains of Kunitz-type protease inhibitors. They are relatively small with a length of about 50 to 60 amino acids and a structure which is a disulfide rich alpha and beta fold. The majority of the sequences having this domain belong to the kunitzbovine pancreatic trypsin inhibitor family which includes bovine pancreatic trypsin inhibitor (BPTI or basic protease inhibitor) and numerous other members such as snake venom basic protease inhibitors like dendrotoxins, mammalian inter-alpha-trypsin inhibitors, trypstatin, a domain found in an alternatively spliced form of Alzheimer's amyloid protein, domains at the C-termini of the alpha-1 and alpha-3 chains of type VI and type VII collagens, tissue factor pathway inhibitor precursor and Kunitz STI protease inhibitor contained in legume seeds.
Dendrotoxins are a group of neurotoxins isolated from the venom of the black mamba (Dendroaspis polyepis polyepis) and the eastern green mamba (Dendroaspis angusticeps) that are selective blockers of particular subtypes of voltage-gated potassium channels in neurons, thereby enhancing the release of acetylcholine at neuromuscular junctions. Because of their high potency and selectivity for potassium channels, dendrotoxins represent useful pharmacological agents for studying the structure and function of these ion channel proteins and for treating human diseases (WO 2007019267). Dentrotoxins are small proteins consisting of a single peptide chain of less than 100 amino acids, generally of about 57-60 amino acids, that folds into the Kunitz structure, i.e., a disulfide rich alpha and beta fold composed of 3 disulfide bridges with the connectivity 1-6, 2-4, 3-5 and arranged to form a twisted two-stranded antiparallel beta sheet followed by an alpha helix (Berndt et al., J. Mol. Biol., 1993, 234, 735-750).